The resolution of an optical microscope is determined by the numerical aperture and wavelength of the objective lens. Generally, it has been considered that the only method for increasing the resolution is to shorten the wavelength or increase the numerical aperture. Meanwhile, there is a demand to observe samples at a resolution higher than that determined by the numerical aperture and wavelength.
One technique for meeting this demand is a technique called “grating super-resolution” indicated by W. Lukosz in “Optical systems with resolving powers exceeding the classical limit. II,” Journal of the Optical Society of America, Vol. 37, No. 7 (1967). In this method, an optical microscope with a high resolution is constructed in which a sample image is spatially modulated by a diffraction grating placed in the vicinity of the object of observation, the spatial frequency component that cannot pass through a focusing optical system that is present between the sample and an image pickup element is conducted to the image pickup element, and this component is demodulated by the diffraction grating in the vicinity of the image pickup element. As Lukosz himself recognizes, this is not realistic; as a more realistic microscope construction, however, there is the microscope construction introduced by (for example) J. T. Frohn et al., in “True optical resolution beyond the Rayleigh limit achieved by standing wave illumination,” PNAS, Vol. 97, No. 13.
With regard to this microscope construction and image processing, for example, a method is described in Japanese Laid-Open Patent Application No. H11-242189 in which an image with a high resolution is obtained by providing a means for modulating the spatial frequency of the illuminating light in the vicinity of the sample that is being observed in the illumination optical system, acquiring a plurality of picked-up images while modulating the spatial frequency, and demodulating the plurality of picked-up images. In the demodulation method described in Japanese Laid-Open Patent Application No. H11-242189, an image is formed by linear calculations on the basis of the plurality of picked-up images.
However, in the demodulation method described in Japanese Laid-Open Patent Application No. H11-242189, no consideration is given to the contribution of the noise that is contained in the picked-up images. Accordingly, there is a possibility that the image that is obtained by demodulation will be erroneous because of the effects of the noise component.
Generally, an optical signal that is output from a photodetector contains noise components of dark current noise, thermal noise, and shot noise. If the image pickup environment is constant, dark current noise is noise which does not depend on other conditions, and which shows a variation in the vicinity of a substantially constant value. Thermal noise is a noise component that depends on the image pickup environment and image pickup conditions. Shot noise is noise which is not greatly affected by the image pickup environment or image pickup conditions, but which greatly depends on the signal strength.
The picked-up image always contains noise components that depend on the signal strength and noise components that do not depend on the signal strength; especially in picked-up images of samples that are not bright, both of these noise components have a relative magnitude that cannot be ignored. Accordingly, without proper removal of the noise components, practically sufficient image recovery is difficult. Consequently, in order to accomplish the pickup of a bright image in microscopic observation at a high magnification, it is necessary to increase the light density on the sample by increasing the intensity of the light source or the like. On the other hand, this leads to the problem of considerable damage to the sample.
In a structured illumination microscopic technique, as is also described in Japanese Laid-Open Patent Application No. H11-242189, signal components in the vicinity of the focal plane can be selectively extracted when samples that have a thickness or height are observed. It is known that blurring from outside the focal depth, which could not be removed in the case of conventional microscopes, can be removed during image processing, and that a high resolution in the direction of the optical axis can also be obtained. However, in cases where samples that have such a thickness are observed, signals from outside the focal depth that are removed in image processing are admixed with the signal prior to signal processing, and also become a source of noise; accordingly, the following problem arises: namely, the noise component that depends on the signal strength becomes relatively larger with respect to the image components following image processing.